Topologically interlocked DNA rings enable bidirectional transcription unleashing of crRNA for low-background and high-sensitivity detection of APE1.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a crucial multifunctional enzyme involved in DNA repair and redox regulation, which has recently emerged as a promising biomarker for various malignancies and a potential therapeutic target for anticancer drug development. However, its clinical application faces significant challenges due to the extremely low abundance of APE1 in early-stage tumors, making conventional detection methods insufficiently sensitive for accurate diagnosis and monitoring. This limitation highlights the urgent need for developing novel ultrasensitive detection technologies to overcome these diagnostic barriers. To address this, we constructed a highly sensitive APE1 detection method with minimal background, leveraging the target initiated bidirectional transcriptional amplification and high-efficiency trans-cleavage activity of Cas12a protein. In this method, two AP sites are initially inserted in the interlocked DNA rings. The presence of APE1 can recognize and excise AP sites to open the interlocked DNA rings, which can initiate the bidirectional strand extension and crRNA transcription reactions to generate large amounts of crRNAs. These crRNAs can further associate with Cas12a proteins to activate their trans-cleavage activities and cleave the quenched fluorescence reporter DNAs to generate enhanced fluorescence signal. The system exhibits a well-defined linear relationship within a range of 1.0 × 10 U/mL to 10.0 U/mL and achieves a detection limit as low as 6.7 × 10 U/mL. Moreover, it performs well in real clinical samples and exhibits effective differentiation between lung cancer patients and healthy individuals. This innovative technology effectively minimizes nonspecific background fluorescence signals when no target is present while greatly expanding the adaptability of Cas12a protein for diverse non-nucleic acid targets. Its high sensitivity and specificity make it a promising analytical tool for APE1 detection, offering significant potential for clinical diagnosis and monitoring of related cancers.